Superoxide dismutase derivatives, a method of producing the same and medicinal uses of the same

ABSTRACT

The invention provides a superoxide dismutase derivative of the general formula 
     
         [SOD][Z] 
    
     wherein [SOD] represents a superoxide dismutase having 1 to 22 or 24 groups each derived from an amino groups by removal of one hydrogen atom in lieu of amino groups; [Z] represents a monovalent copolymer group, constituting units of which are a group of the formula ##STR1## wherein R 1 , R 2 , R 3  and R 4  each represents a hydrogen atom or a residue derived by a removal of a hydroxyl group from an alkanol of 1 to 8 carbon atoms, an ethylene glycol monoalkyl ether containing an alkyl moiety of 1 to 4 carbon atoms or a glycerin dialkyl ether containing alkyl moieties of 1 to 4 carbon atoms, provided that either R 1  or R 2  and either R 3  or R 4  each represents a hydrogen atom, and a residue derived from the group of the above-mentioned formula by removal of OR 1 , OR 2 , OR 3  or OR 4  group from one of its COOR 1 , COOR 2 , COOR 3  and COOR 4  groups (where the bond on the carbon atom of the carbonyl group is attached to [SOD]), said monovalent copolymer group having an average molecular weight of 500 to 200,000; and n represents an integer of 1 to 22 or 24 corresponding to the number of said groups each derived from an amino group by removal of one hydrogen atom in said [SOD], or a pharmaceutically acceptable salt thereof. There also is provided a method for producing the above derivative and salt. The invention is further directed to medicinal uses and pharmaceutical compositions for oral administration.

This application is a divisional of application Ser. No. 07/199,809,filed May 27, 1988.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to superoxide dismutase derivatives, amethod of producing the same, medicinal uses for said derivatives, andcompositions containing said derivatives for oral administration.

2. Description of Prior Art

It is known that superoxide dismutase (hereinafter referred to brieflyas SOD) is omnipresent in the living tissues of animals, plants andmicroorganisms and is an enzyme decomposing superoxide which is harmfulto living organisms. Recently, attempts have been made to utilizeisolated SOD as an antiinflammatory agent (FARUMASHIA, 17, 411, 1981 andCurrent Therapeutic Research, 16, 706, 1974).

It is also known that when SOD is administered intravenously, its plasmahalf-life is as short as 4 to 6 minutes and that the administered SOD israpidly excreted. For prolonging the plasma half-life of SOD, an attempthas been made to modify SOD with Ficoll, polyethylene glycol, ratalbumin or inulin [Japanese Published Unexamined Patent Application(Kokai Tokkyo Koho) No. 32826/83].

It has been reported that the SOD activity of Ficoll- or polyethyleneglycol-modified SOD is drastically lower than that of unmodified SOD,while rat albumin-modified SOD is antigenic. It has also been found thatinulin-modified SOD is considerably inferior to SOD in enzymaticactivity.

It is an object of the invention to provide a novel superoxide dismutasederivative having a remarkably extended plasma half-life as comparedwith SOD while retaining the enzymatic activity of SOD substantiallyintact.

It is another object of the invention to provide a novel superoxidedismutase derivative which has pharmacological actions such asantiinflammatory action and so on and is safe.

It is a still another object of the invention to provide a method ofproducing said superoxide dismutase derivative.

It is a further object of the invention to provide medicinal uses, forexample as an antiinflammatory drug, of said superoxide dismutasederivative.

It is still another object of the invention to provide compositionssuitable for oral administration of said superoxide dismutasederivative.

These objects as well as other objects and advantages of the presentinvention will become apparent to those skilled in the art from thefollowing detailed description.

SUMMARY OF THE INVENTION

The present invention provides a superoxide dismutase derivative of thegeneral formula

    [SOD][Z].sub.n

wherein [SOD] represents a superoxide dismutase having 1 to 22 or 24groups each derived from an amino group by removal of one hydrogen atomin lieu of amino groups; [Z] represents a monovalent copolymer group,constituting units of which are a group of the formula ##STR2## whereinR¹, R², R³ and R⁴ each represents a hydrogen atom or a residue derivedby removal of a hydroxyl group from an alkanol of 1 to 8 carbon atoms,an ethylene glycol monoalkyl ether containing an alkyl moiety of 1 to 4carbon atoms or a glycerin dialkyl ether containing alkyl moieties of 1to 4 carbon atoms, provided that either R¹ or R² and either R³ or R⁴each represents a hydrogen atom, and a residue derived from the group ofthe above-mentioned formula by removal of OR¹, OR², OR³ or OR⁴ groupfrom one of its COOR¹, COOR², COOR³ and COOR⁴ groups (where the bond onthe carbon atom of the carbonyl group is attached to [SOD]), saidmonovalent copolymer group having an average molecular weight of 500 to200,000; and n represents an integer of 1 to 22 or 24 corresponding tothe number of said groups each derived from an amino group by removal ofone hydrogen atom in said [SOD], or a phamaceutically acceptable saltthereof.

The present invention further provides a method of producing the abovesuperoxide dismutase derivative (hereinafter referred to briefly as SOD)or the pharmaceutically acceptable salt thereof, which comprisesreacting superoxide dismutase with a copolymer, constituent units ofwhich are: ##STR3## wherein R¹, R², R³ and R⁴ each represents a hydrogenatom or a residue derived by removal of a hydroxyl group from an alkanolof 1 to 8 carbon atoms, an ethylene glycol monoalkyl ether containing analkyl moiety of 1 to 4 carbon atoms or a glycerin dialkyl ethercontaining alkyl moieties of 1 to 4 carbon atoms, provided that eitherR¹ or R² and either R³ or R⁴ each represents a hydrogen atom, and (b) agroup selected from the class consisting of a group of the formula##STR4## wherein R¹ and R² are respectively as defined above, a group ofthe formula ##STR5## wherein R³ and R⁴ are respectively as defined aboveand a group of the formula ##STR6## said copolymer having an averagemolecular weight of 500 to 200,000 (hereinafter referred to briefly asthe copolymer) in the presence of a basic aqueous solution of pH 7-11.

The present invention provides, in another aspect, a pharmaceuticalcomposition such as an antiinflammatory agent which contains said SODderivative or a salt thereof as an active ingredient and a method fortreatment of inflammatory and other diseases which comprisesadministering an effective amount of said SOD derivative or saltthereof.

In a further aspect, the present invention provides a pharmaceuticalcomposition for oral administration which comprises said SOD derivativeor a salt thereof and a medium/higher fatty acid glyceride, and apharmaceutical composition for oral administration which comprises saidSOD derivative or a salt thereof, a medium/higher fatty acid glyceride,and an amphiphilic agent and/or a lower alkanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of the SOD derivative and SOD on influenza virusinfected mice.

FIG. 2 is an ultraviolet absorption spectrum of the SOD derivativeprepared in Example of Synthesis 1;

FIG. 3 is an infrared absorption spectrum of the SOD derivative preparedin Example of Synthesis 2;

FIG. 4 (i) is a densitogram as measured by polyacrylamideelectrophoresis of the starting material SOD used in Example ofSynthesis 2, and

FIG. 4 (ii) is a densitogram as measured by polyacrylamideelectrophoresis of the SOD derivative prepared in Example of Synthesis2.

DETAILED DESCRIPTION OF THE INVENTION (a) Method for Production of theSOD Derivative (a-1) Reaction of SOD with the Copolymer

The reaction of SOD with the copolymer is generally conducted bydissolving SOD in an aqueous solution of a salt such as sodiumcarbonate, sodium hydrogen carbonate, sodium acetate, sodium phosphate,etc. and adding the copolymer, either in powder form or as dissolved inan organic solvent such as dimethyl sulfoxide, to the resultingsolution. It is necessary that the pH of the solution be maintained inthe range of 7 to 11 during the reaction. If the pH is below 7, thesolubility of the copolymer is decreased to interfere with progress ofthe reaction. If the pH is higher than 11, the SOD is inactivated sothat an effective SOD derivative cannot be obtained. The reactiontemperature is preferably about 3° to 50° C. and, for still betterresults, in the range of 3° to 40° C. While the reaction time isdependent on the reaction temperature and the method of addition of thecopolymer, it is generally in the range of 10 minutes to 3 hours. Theproportion of the copolymer is in the range of about 0.5 to 30 moles to1 mole of SOD. By varying this ratio, the number of molecules of thecopolymer to be attached to SOD can be adjusted.

The resulting reaction mixture contains not only the SOD derivative butalso the unreacted SOD and copolymer, etc. Therefore, the reactionmixture is filtered and the filtrate is subjected to gel filtration. Theresulting filtrate containing the SOD derivative is subjected, ifnecessary, to hydrophobic chromatography and to ultrafiltration. Theconcentrate thus obtained is freeze-dried to give the SOD derivative asa solid product.

As the result of the reaction described above, the amino groups of SODare bound to the maleic anhydride moieties of the copolymer to give theSOD derivative. For example, human type SOD contains 22 (humanerythrocyte SOD or genetically engineered human-type SOD produced byyeast) or 24 (genetically engineered human-type SOD produced byEscherichia coli) amino groups per molecule. In the above reaction, acertain one of said amino groups of SOD reacts with one of the maleicanhydride moieties of the copolymer to ultimately give a SOD derivativecontaining 1 to 22 or 24 molecules of the copolymer per molecule of theSOD. The starting material copolymer generally contains maleic anhydriderings as described later. When one of these maleic anhydride rings isbound to an amino group of SOD, the remaining maleic anhydride ring doesnot react appreciably with another amino group but tends to react withwater, giving rise to a maleic acid-derived group of the formula##STR7## It is possible that one molecule of the copolymer reacts with aplurality of molecules of SOD to give a byproduct compound in which aplurality of maleic anhydride rings in the copolymer molecule are boundto amino groups of the respective SOD molecules but it is notobjectionable to use the SOD derivative including a small proportion ofsuch byproduct in the practice of the invention. However, inconsideration of the fact that any active ingredient of a pharmaceuticalproduct is desirably a compound having a single chemical structure, itis preferable to purify the byproduct-rich SOD derivative by a suitableprocedure such as gel filtration so as to remove the byproduct.Furthermore, it should be understood that the SOD derivative obtained bythe above reaction is actually a mixture of compounds containingdifferent numbers of molecules of the copolymer, that is to say thenumber of molecules of the copolymer bound to SOD is not uniform overthe respective SOD derivative compounds. Therefore, in the above generalformula representing the SOD derivative, n means the average number ofmolecules of the copolymer bound to one molecule of SOD. However, whenit is desirable to obtain the SOD derivative in which the number ofmolecules of the copolymer bound to SOD is uniform, the SOD derivativeobtained by the above method may be further subjected to a suitablefractional purification procedure such as gel filtration to give thedesired SOD derivative.

(a-2) The Starting Material SOD

The SOD used as a starting material may be one extracted from the livingtissue of an animal (man, cattle, etc.), plant or microorganism by theconventional procedure or one obtained by a genetic engineering method.The chemical structure (coordinating metal, molecular weight, amino acidsequence, etc.) of SOD has by now been elucidated fairly well. Thus, SODhas been classified into the three major types of Fe- coordinating SOD,Mn-coordinating SOD and Cu.Zn-coordinating SOD and, depending on thebiological tissue in which it occurs, has a molecular weight in therange of 30,000 to 80,000. While the amino acid sequence of SOD alsovaries somewhat with different biological tissues in which it iscontained, detailed information on this subject can be obtained from thefollowing literature: Yoshihiko Oyanagi: Superoxide and Medicine, 74-90(Kyoritsu Shuppan, May 25, 1981); Journal of Biological Chemistry, 246,2875-2880 (1971); and 250, 6107-6112 (1975); Proceedings of the NationalAcademy of Sciences, 70, 3725-3729 (1973); Archives of Biochemistry andBiophysics, 179, 243-256 (1977) and so on. The starting material SOD ispreferably a human-type Cu.Zn-coordinating SOD. The bovine- andequine-type Cu.Zn-coordinating SODs are also suitable for the purposesof the invention. This kind of SOD has a molecular weight of about33,000 and contains 20, 22 or 24 amino groups in its molecule. The humantype SOD can, for instance, be isolated by subjecting human blood toheat treatment, ion exchange and gel filtration in succession or by agenetic engineering method.

(a-3) The Starting Material Copolymer

The copolymer as another starting material can be obtained by subjectinga divinyl ether-maleic anhydride copolymer to partial hydrolysis (degreeof hydrolysis of maleic anhydride rings: 30-90%) or subjecting saidcopolymer to partial half-esterification (degree of half-esterificationof maleic anhydride rings: 30-90%). As examples of the ester, there maybe mentioned methyl ester, ethyl ester, propyl ester, n-butyl ester,n-pentyl ester, isopentyl ester, n-hexyl ester, n-heptyl ester, n-octylester, methoxyethyl ester, ethoxyethyl ester, propoxyethyl ester,2-butoxyethyl ester, 1,3-dimethoxy-2-propyl ester,2,3-dimethoxy-1-propyl ester, 1,3-diethoxy-2-propyl ester,2-ethoxy-3-methoxy-1-propyl ester, 1,3-dipropoxy-2-propyl ester,1,3-dibutoxy-2-propyl ester, benzyl ester and so on.

The starting material copoymer has, as aforesaid, a weight averagemolecular weight in the range of 500 to 200,000. In view of the transferkinetics of the final SOD derivative to the affected site in vivo, theweight average molecular weight of the copolymer is preferably not over10,000. There is no particular limitation on the molecular weightdistribution of the copolymer. Thus, the copolymer (whose weight averagemolecular weight/number average molecular weight ratio is about 2.0 orhigher) synthesized by the radical copolymerization of divinyl etherwith maleic anhydride and directly subjected to partial hydrolysis orpartial half-esterification without prior fractionation or afterfractionation for narrowing its molecular weight distribution can besuccessfully employed as the starting material copolymer.

(b) The Pharmaceutically Acceptable Salt of SOD Derivative, and aProcess for Preparation of the Salt.

As examples of the aforesaid pharmaceutically acceptable salt of SODderivative, there may be mentioned the salts of the derivative withalkali metals such as sodium, potassium, etc., salts with alkaline earthmetals such as magnesium, calcium, barium, etc., ammonium salt, andsalts with tertiary amines such as pyridine, triethylamine,tri-n-butylamine and so on.

The pharmaceutically acceptable salt of SOD derivative can be easilyprepared from the SOD derivative and the desired kind of base by theroutine salt forming reaction.

(c) Compositions for Oral Administration

The medium/higher fatty acid glyceride which is used for preparation ofthe composition for oral administration according to the invention isthe mono-, di- or triglyceride of a saturated or unsaturated fatty acidcontaining 6 to 20 carbon atoms. Representative examples of such fattyacid glyceride include the mono-, di- and triglycerides of caprylicacid, capric acid, lauric acid, myristic acid, palmitic acid, oleicacid, linoleic acid, linolenic acid or the like. These fatty acidglycerides can be used singly or in combination.

The fatty acid glyceride may be a naturally-occuring compound or asynthetic or semi-synthetic compound. Usually, it is expedient to employa natural vegetable oil. The vegetable oils which can be employed withadvantage in the practice of the invention include, among others, oliveoil (oleic acid 70-80%, linolic acid 4-12%, palmitic acid 7-15%), maizeoil (linolic acid 40-60%, palmitic acid 25-45%), sesame oil (oleic acid35-46%, linolic acid 35-48%), camellia oil, coconut oil (lauric acid45-52%, capric acid 4-12%, caprylic acid 6-10%) and palm oil. Commercialproducts can be used as such. Thus, for example, commercially availablemedium fatty acid triglycerides such as Panasate® 875, 810 and 800(Nippon Oil and Fats Co., Ltd.; caprylic acid content 10-100%), ODD®(Nissin Seiyu K.K.; caprylic acid content 67%), etc. can be utilized. Asan example of commercial medium fatty acid monoglyceride, there may bementioned Homoteks®PT (Kao Corporation; capric acid content 60%). As anexample of medium fatty acid mono- and di- glyceride mixture, there maybe mentioned Witafrol® (Dinamit Novel Corporation). As for higher fattyacid triglyceride, commercial edible oils such as olive oil from WakoPure Chemical Industries Ltd. and linolic acid from Nippon Oil and FatsCo., Ltd. can be utilized.

The aforesaid amphiphilic agent is a non-toxic agent having bothhydrophilicity and lipophilicity. Typical examples of such amphiphilicagent include natural amphoteric surfactants, polyglycerin fatty acidester, polyoxyethylene-sorbitan fatty acid ester (Tween series),sorbitan fatty acid ester (Span series) and polyethylene glycol.Preferred amphoteric surfactants are soybean phospholipid, yolk lecithinand their related substances, such as commercial phosphatidylcholine,yolk lecithin, soybean lecithin, phosphatidylethanolamine, etc.available from Nippon Oil and Fats Co., Ltd. The polyglycerin fatty acidester may for example be Unigli® (Nippon Oil and Fats Co., Ltd). As anexample of the polyoxyethylene sorbitan fatty acid ester, there may bementioned Tween®20 (Wako Pure Chemical Industries, Ltd.). The sorbitanfatty acid ester may for example be Span®20 (Wako Pure ChemicalIndustries, Ltd.), while the polyethylene glycol may be PEG 6000, forinstance. Aside from the above, anionic surfactants such as sodiumlaurylsulfate and cationic surfactants such as benzalkonium chloride,benzethonium chloride, Eison® (Nelson Research & Development Co.,U.S.A.), etc. can also be employed

The aforesaid alkanol may for example be ethanol, propanol, isopropylalcohol, butanol or the like.

In the production of a composition according to the invention, alyophilizate from aqueous solution of SOD derivative or itspharmaceutically acceptable salt (hereinafter referred to collectivelyas SOD derivative) adjusted to an appropriate pH is uniformly dispersedin said fatty acid glyceride containing, or not containing, saidamphiphilic agent and/or lower alkanol or, alternatively, a mixture of asolution of SOD derivative in aqueous ammonium carbonate solution and anaqueous solution of said amphiphilic agent and/or lower alkanol islyophilized and the lyophilizate is uniformly dispersed in themedium/higher fatty acid glyceride.

The proportion of said fatty acid glyceride is about 0.1 to 100 ml permg of the SOD derivative and preferably about 0.5 to 5 ml on the samebasis. The addition of said amphiphilic agent and/or lower alkanol isoptional but these agents contribute to enhanced wettability with theoil and increased dispersibility or solubility therein so as to give astable composition with an additional effect of enhanced absorptionafter oral administration. The proper level of addition of saidamphiphilic agent varies with different species thereof. Generally,however, with respect to 1 mg of SOD derivative, it is appropriate toemploy 0.01 to 0.1 ml when the amphiphilic agent is a liquid or 0.05 to5 mg when it is a solid agent. The level of addition of said loweralkanol may be about 1 to 15 weight percent based on the total weight ofthe composition. The addition of such lower alkanol leads to an improvedhomogeneity of the solution.

The composition according to the invention is a clear liquid which isstable physically and chemically. Thus, when the composition wascentrifuged at 5000 rpm for 15 minutes at room temperature or allowed tostand at 37° C. for 1 month, no sedimentation was observed. Furthermore,when stored in the dark at 4° C. or at room temperature, the compositionof the invention showed no macroscopic change for at least 3 months. Inaddition, no macroscopic change was observed even after the compositionwas subjected to 10 cycles of temperature variation from 4° C. to roomtemperature at 24-hour intervals and its titer remained unaffected.

(d) Pharmacological Profile of the SOD Derivative

The SOD derivative according to the invention features a remarkablyextended plasma half-life as compared with SOD while retaining theenzymatic activity of SOD substantially intact and is, furthermore, lowin toxicity. Therefore, the SOD derivative of the invention is of valueas an antiinflammatory agent and an agent for ischemic diseases.

The following animal experiments illustrate the effects of the SODderivative and composition of the invention.

(I) ANTIINFLAMMATORY AND ANTIVIRUS EXPERIMENTS (1) Effect on PulmonaryConsolidation (Inflammatory Sclerosis) in Influenza-infected Mice Method

ddY mice aged 5-6 weeks and weighing about 22 g were infected withinfluenza virus [A2/Kumamoto (H₂ N₂)] in a dose of 100 times the LD₅₀ inmice by inhalation of a virus aerosol and, then, the test agent wasorally administered in predetermined doses once a day for 6 consecutivedays from the day of infection. The mice were used in groups of 30. Themice were serially sacrificed 3, 4 and 5 days after infection and themouse pulmonary consolidation scores were determined by the method ofHorsfall (Journal of Experimental Medicine, 95, 135-145, 1952).

The results are shown in Table 1. The consolidation scores in the tablewere determined as follows. Physiological saline was injected directlyinto the mouse heart to wash the lung thoroughly and the lung was thenobserved. The lesion was evaluated using the following scoring scale.

    ______________________________________                  Percentage of consolidation    Consolidation score                  in lung field    ______________________________________    0             0    0.5           12.5    1.0           25    1.5           37.5    2.0           50    2.5           62.5    3.0           75    3.5           87.5    4.0           100    5.0           Death due to infection    ______________________________________

                  TABLE 1    ______________________________________                    Time (in days) after in-                    fection with influenza virus    Test agent          3        4      5    ______________________________________    Physiological saline (control)                        0.67     0.89   1.33    ODO ® alone (control)                        0.61     0.91   1.39    SOD derivative of Example                        0.0      0.17   0.33    of Synthesis 1*    (2,000 units/mg)    Composition of Formulation                        0.0      0.11   0.15    Example 6**    (2,000 units/mg)    ______________________________________      *An aqueous solution      **An oily composition

(2) Effect on Virus Infected Mice Method

ddY mice aged 5-6 weeks and weighing about 22 g were infected withinfluenza virus [A₂ /Kumamoto (H₂ N₂)] in a dose of 2 times the LD₅₀ inmice by inhalation of a virus aerosol and, then, the SOD derivative ofExample of Synthesis 1 (2000 units/mg) or bovine erythrocyte SOD (2000units/m9) was intravenously administered in a dose of 200 units/mouseonce a day for 4 consecutive days from the 5th day after infection.

FIG. 1 shows % survival plotted against days after infection. The %survival was 95% in the SOD derivative group, and 0% in the SOD groupand in the control group (infected and not treated mice). In a furthertest, the SOD was administered in a dose of 2000 units/mouse in the samemanner as above, and only a slight effect was observed.

It is evident from the results that the SOD derivative has an excellenteffect on influenza virus infected mice.

(3) Effect on Albumin Leakage at the Site of Burn Method

Evan's blue was dissolved in physiological saline at a finalconcentration of 0.2 weight % and 0.18 ml (10 mg/kg) of the solution wasadministered intravenously to ddY mice aged 8-10 weeks and weighingabout 30 g. The mice were used in groups of 10. After administration,the head (12 mm diameter) of an iron nail preheated in a water bath at70° C. was pressed against the abdomen of the mouse to cause a burn andthe test agent was orally administered immediately. Two hours afteradministration of the test agent, the mice were sacrificed and the skinat the site of burn was excised and immersed in formamide at 60° C. for48 hours. The concentration of extracted Evans' blue was determinedbased on the absorbance at 620 nm. The concentration of the Evans'blue-albumin complex was estimated from the data and the % inhibition ofalbumin leakage at the site of burn due to the test agent wascalculated.

The results are shown in Table 2.

                  TABLE 2    ______________________________________                           Concentration                           of Evans' blue-                Dosage     albumin complex                                        Percent    Test agent  (units/mouse)                           (μg/g protein)                                        Inhibition    ______________________________________    Physiological saline                 0         82.0         --    (control)    ODO ® alone                 0         87.3           0    (control)    SOD derivative of                400        41.3         49.6    Example of    Synthesis 1*    (2,000 units/mg)    Composition of                400        38.1         53.5    Formulation    Example 6**    (2,000 units/mg)    ______________________________________      *An aqueous solution      **An oily composition

The above experiment was repeated except that the mice were sacrificed 6hours after administration. Then, in the same manner as above, theconcentration of Evans' blue-albumin complex was determined. The %inhibition of albumin leakage at the site of burn was 0% in the ODO®alone group, 50.0% in the SOD derivative (Example of Synthesis 1) group,and 56.5% in the SOD composition (Formulation Example 6) group.

The same result was obtained with the SOD derivative according toExample of Synthesis 2.

It will be apparent from the above results that the SOD derivative andcomposition of the invention significantly inhibited the albumin leakageat the site of burn as compared with the control groups.

(II) EXPERIMENT OF THE BLOOD TRANSFER OF THE SOD DERIVATIVE COMPOSITIONAFTER ORAL ADMINISTRATION

The blood SOD derivative concentration was determined by radioactivityassay. The experimental procedures and results are given below.

Preparation of ¹⁴ C Glycine-labeled SOD Derivative

In 1.5 ml of distilled water was dissolved 17.7 mg of the SOD derivativeaccording to Example of Synthesis 1, followed by addition of 18.2 mg ofwater-soluble carbodiimide. After 5 minutes, 0.13 mg (0.5 ml aqueoussolution) of ¹⁴ C glycine (New England Nuclear, U.S.A.; 113.0 mCi/mmole)was added. The pH of the mixture was adjusted to about 6 with 1M aqueoussodium hydrogen carbonate solution and the reaction was allowed toproceed under gentle stirring at room temperature in the dark for 1hour. Then, 1.0 ml of 1M acetate buffer (pH 6.0) was added so as toterminate the reaction and the reaction mixture was desalted with acolumn (2.3×10 cm) of Sephadex®G-25 (Pharmacia F.C.) and lyophilized.The procedure gave 15.5 mg of ¹⁴ C glycine-labeled SOD derivative with aspecific radioactivity of 10.7 μCi/mg. This product was a substantiallyuniform product mainly containing one glycine unit per molecule of SODderivative. This product was a stable compound in which glycine had beenbound by amide linkage to the carboxyl group of the SOD derivative.

(2) Oral Administration Experiment in Animals

Using the ¹⁴ C glycine-labeled SOD derivative, a composition wasprepared in accordance with the method described hereinafter inFormulation Example 2 and using a gastric tube, 0.2 ml (4.26 μCi) of thecomposition was orally administered to male ddY mice aged 8 weeks. Themice were sacrificed serially at 3, 7 and 24 hours after administrationand the radioactivity levels in the plasma and major organs weredetermined. The radioactivity was expressed in dpm per 1.0 g of tissue.The results are shown in Table 3.

                  TABLE 3    ______________________________________    Time course of tissue concentration    of .sup.14 C glycine-labeled SOD derivative    after oral administration    Tissue  After 3 hr    After 7 hr                                    After 24 hr    ______________________________________    Plasma  9304           7912      9908    Liver   32048         50224     90027    Kidney  12054         28465     62873    Spleen  5912          10392     22644    Muscle  2490           3452     12200    ______________________________________

The data in Table 3 show that the composition containing the SODderivative prepared in Example of Synthesis 1 is efficiently transferredto the blood and various organs and tissues. The same result was alsoobtained with a similar composition containing the SOD derivativeobtained in Example of Synthesis 2.

Thus, the SOD derivative of the invention, when formulated into thecomposition of the invention and administered orally, producesparticularly remarkable antiinflammatory effects.

For clinical application of the above composition to humans, thecomposition can be further processed into suitable dosage forms such assoft capsules, capsules, tablets, granules, liquids, suppositories, etc.and administered to patients.

Such preparations are generally administered in a dose of 0.1 to 100 mg(as SOD derivative) 1 to 5 times a day, either every day or every otherday. The dosage is adjusted according to the patient's condition andother factors.

(e) EXAMPLES

The following synthesis and formulation examples are furtherillustrative of the invention, it being to be understood, however, thatthe invention is by no means limited to these specific examples.

EXAMPLE OF SYNTHESIS 1

In 10 ml of 0.1M aqueous sodium hydrogen carbonate solution (pH 8.0) wasdissolved 95 mg of bovine erythrocyte SOD (Sigma) with stirring at 4° C.To this solution was gradually added 17 mg of a solid powder of partialhydrolyzate (degree of hydrolysis=about 50%) of a divinyl ether-maleicanhydride copolymer (divinyl ether to maleic anhydride mole ratio=1:2,Mw=5,600, molecular weight distribution:Mw/Mn=about 1.5) (Mw=weightaverage molecular weight, Mn =number average molecular weight) and thereaction was conducted for about 1.0 hour. The reaction mixture waspoured into a column (4.2×80 cm) of Sephadex®G-50 (Pharmacia F.C.) forgel filtration and elution was carried out with distilled water. Theeluate was monitored by the absorbance method at 280 nm and 220 nm andthe fraction containing the unreacted divinyl ether-maleic anhydridecopolymer partial hydrolyzate was discarded. The remaining fraction waslyophilized to give the SOD derivative as a white power.

The ultraviolet absorption spectrum of the SOD derivative thus obtainedwas measured in phosphate buffer at the concentration of 1 mg/ml and pH7.4. The UV absorption spectrum of this product is shown in FIG. 2.

The bovine erythrocyte SOD used above contained 20 amino groups permolecule In order to confirm that these amino groups had reacted withthe partial hydrolyzate of divinyl ether-maleic anhydride copolymer, theassay of residual amino groups was performed using sodiumtrinitrobenzenesulfonate (TNBS). It was found that under the aboveconditions of reaction, about 22 mole % of the amino groups of bovineerythrocyte SOD had reacted so that an average of 4 to 5 molecules ofthe divinyl ether-maleic anhydride copolymer partial hydrolyzate werebound to each molecule of SOD.

When the enzymatic activity of the SOD derivative was determined by thepyrogallol auto-oxidation method described in European Journal ofBiochemistry, 47, 469-474, 1974, the SOD derivative was found to retain45% of the enzymatic activity of the original SOD.

EXAMPLE OF SYNTHESIS 2 (a) Synthesis of Partially Half-esterifiedDivinyl Ether-maleic Anhydride Copolymer

A 100 ml (approx.) ampule with a magnetic stirrer was charged with 2.0 gof divinyl ether-maleic anhydride copolymer (divinyl ether to maleicanhydride mole ratio =1:2, Mw=5600, molecular weight distribution:Mw/Mn=1.5), 0.88 g of n-butanol, 40 mg of anhydrous lithium acetate and60 ml of tetrahydrofuran, followed by sealing. The contents of theampule were heated with stirring at 55° C. for 20 hours. An aliquot ofthe reaction mixture was sampled and the unreacted n-butanol in thereaction mixture was estimated by gas chromatography usingethyl-cellosolve as an internal standard. From the reaction rate ofcharged n-butanol, the degree of conversion of the maleic anhydride ringof the copolymer to the n-butyl half-ester was calculated to be 43.3mole %. The reaction mixture was then concentrated under reducedpressure and the concentrate was added dropwise into 500 ml of n-hexanefor reprecipitation. The resulting precipitate was recovered and driedat room temperature overnight to give 1.13 g of the desired n-butylhalf-esterified divinyl ethermaleic anhydride copolymer.

(b) Synthesis of the SOD Derivative by Reaction of N-butylHalf-esterified Divinyl Ether-maleic Anhydride Copolymer with SOD

In 1.8 ml of isotonic phosphate buffer (pH 7.04) were dissolved 200 μlof aqueous human erythrocyte SOD solution (62.8 mg/ml) and 170 mg ofsodium hydrogen carbonate. To this solution was gradually added 64 mg ofthe n-butyl half-esterified divinyl ether-maleic anhydride copolymerprepared as above (a) with stirring at room temperature. The reactionwas conducted for 2 hours and, then, allowed to stand at 4° C.overnight. The reaction mixture was filtered and the filtrate wassubjected to gel filtration on Sephadex®G-75 (Pharmacia F.C.) using 10mM aqueous ammonium bicarbonate as an eluent. The fraction containingthe SOD derivative was lyophilized to give 8.52 mg of the SOD derivativeas a white powder. The infrared absorption spectrum (KBr disk) of thisproduct is shown in FIG. 3. The densitogram of the starting material SODas determined by polyacrylamide electrophoresis (native PAGE) is shownin FIG. 4 (i) and that of the SOD derivative in FIG. 4 (ii). In thedrawings, a represents SOD and b the SOD derivative.

The SOD derivative used in the following formulation examples wasobtained according to Example of Synthesis 1. Comparable compositionsare also obtainable by using other SOD derivatives according to thisinvention.

FORMULATION METHOD 1

The SOD derivative prepared in Example of Synthesis 1 is dissolved indistilled water (10 mg/ml) under ice cooling. The solution is adjustedto pH about 3.0 by dropwise addition of 0.5 M acetic acid andlyophilized. To the freeze-dried SOD derivative powder was added amedium/higher fatty acid glyceride supplemented with an amphiphilicagent and/or a lower alkanol and the mixture is shaken until amacroscopically uniform dispersion is obtained. The shaking treatment iscarried out using the TOMY UR-150P chip-type supersonic wave generator(Tomy Seiki).

The duration of this treatment is not more than 30 seconds.

FORMULATION METHOD 2

A predetermined amount of an amphiphilic agent is added to a givenquantity of distilled water and after addition of a lower alkanol asrequired, ultrasonic treatment is carried out to prepare a solution.Then, a solution (4 mg/ml) of SOD derivative powder (Example ofSynthesis 1) in 0.02% aqueous ammonium carbonate solution, preparedunder ice cooling, is added to the above solution in equal volumes andthe mixture is lyophilized. To the resulting freeze-dried powder isadded the medium/higher fatty acid glyceride, followed by addition ofthe lower alkanol as required. The mixture is ultrasonicated in anice-water bath for 30 seconds.

The above procedure gave compositions of the invention as shown in Table4.

                                      TABLE 4    __________________________________________________________________________         SOD derivative         prepared in    Formula-         Example of                 Medium/higher   Amphiphilic agent and/or                                                   Method of    tion No.         Synthesis 1                 fatty acid glyceride                                 lower alkanol     preparation    __________________________________________________________________________     1   1 mg    Panasate ®875                            1  ml                                 Phosphatidylcholine                                              0.1                                                 g 1     2   1 mg    Panasate ®810                            0.95                               ml                                 Unigli ® GO-206                                              0.05                                                 ml                                                   2     3   1 mg    Panasate ®800                            0.25                               ml                                 Yolk lecithin                                              0.1                                                 g 1                 Olive oil  0.75                               ml     4   1 mg    ODO ®  0.25                               ml                                 Soybean lecithin                                              0.1                                                 g 1                 Linolic acid                            0.75                               ml     5   1 mg    Panasate ®875                            0.95                               ml                                 Phosphatidylethanolamine                                              2  mg                                                   2                                 Unigli ® GO-206                                              0.05                                                 ml     6   1 mg    Panasate ®875                            0.8                               ml                                 Phosphatidylcholine                                              2  mg                                                   2                 Homoteks ®PT                            0.2                               ml     7   1 mg    10% ethanol-ODO ®                            1  ml                                 Yolk lecithin                                              2  mg                                                   2     8   1 mg    Panasate ®810                            0.95                               ml                                 Phosphatidylethanolamine                                              0.1                                                 g 2                                 Unigli ®GO-206                                              0.05                                                 ml     9   1 mg    ODO ®  1  ml                                 Soybean lecithin                                              0.1                                                 g 2                                 PEG 6000     0.1                                                 g    10   1 mg    Panasate ®800                            0.9                               ml                                 Tween ® 20                                              0.05                                                 ml                                                   2                                 Span ®20 0.05                                                 ml    11   1 mg    ODO ®  1  ml                                 --                1    12   1 mg    Panasate ®800                            1  ml                                 --                1    13   1 mg    Panasate ®810                            1  ml                                 --                1    14   1 mg    Linolic acid                            1  ml                                 --                1    15   1 mg    Olive oil  1  ml                                 --                1    16   1 mg    Panasate ®875                            1  ml                                 --                1    17   1 mg    Edible oil 1  ml                                 --                1                 [Nisshin Seiyu K.K.]    18   1 mg    ODO ®  0.5                               ml                                 --                1                 Edible oil 0.5                               ml                 [Nisshin Seiyu K.K.]    19   1 mg    ODO ®  0.5                               ml                                 Phosphatidylcholine                                              1  mg                                                   2                 Edible oil 0.5                               ml                 [Nisshin Seiyu K.K.]    20   1 mg    ODO ®  0.95                               ml                                 Eison ®  0.05                                                 ml                                                   2    21   0.1 mg  ODO ®  1  ml                                 --                 1    22   10 mg   ODO ®  1  ml                                 --                1    23   20 mg   ODO ®  1  ml                                 --                1    24   30 mg   ODO ®  1  ml                                 --                1    25   1 mg    Panasate ®875                            1  ml                                 Unigli ® GO-206                                              0.05                                                 ml                                                   1                                 Ethanol      0.15                                                 ml    26   1 mg    Panasate ®875                            1  ml                                 Unigli ®GO-206                                              0.05                                                 ml                                                   2                                 Ethanol      0.15                                                 ml    __________________________________________________________________________

What is claimed is:
 1. A method of treating an inflammatory diseasewhich comprises administering to a patient with the disease atherapeutically effective amount of a superoxide dismutase derivative ofthe general formula

    [SOD][Z].sub.n

wherein [SOD] represents a superoxide dismutase having 1 to 22 or 24groups each derived from an amino group by removal of one hydrogen atomin lieu of amino groups; [Z] represents a monovalent copolymer group,constituting units of which are a group of the formula ##STR8## whereinR¹, R², R³ and R⁴ each represents a hydrogen atom or a residue groupderived by removal of a hydroxyl from an alkanol of 1 to 8 carbon atoms,an ethylene glycol monoalkyl ether containing an alkyl moiety of 1 to 4carbon atoms or a glycerin dialkyl ether containing alkyl moieties of 1to 4 carbon atoms, provided that either R¹ or R² and either R³ or R⁴each represents a hydrogen atom, and a residue derived from the group ofthe above-mentioned formula by removal of OR¹, OR², OR³ or OR⁴ groupfrom one of its COOR¹, COOR², COOR³ and COOR⁴ groups (where the bond onthe carbon atom of the carbonyl group is attached to [SOD]), saidmonovalent copolymer group having an average molecular weight of 500 to200,000; and n represents an integer of 1 to 22 or 24 corresponding tothe number of said groups each derived from an amino group by removal ofone hydrogen atom in said [SOD], or a pharmaceutically acceptable saltthereof.
 2. A pharmaceutically composition for oral administrationcomprising a superoxide dismutase derivative of the general formula

    [SOD][Z].sub.n

wherein [SOD] represents a superoxide dismutase having 1 to 22 or 24groups each derived from an amino group by removal of one hydrogen atomin lieu of amino groups; [Z] represents a monovalent copolymer group,constituting units of which are a group of the formula ##STR9## whereinR¹, R², R³ and R⁴ each represents a hydrogen atom or a residue derivedby removal of a hydroxyl group from an alkanol of 1 to 8 carbon atoms,an ethylene glycol monoalkyl ether containing an alkyl moiety of 1 to 4carbon atoms or a glycerin dialkyl ether containing alkyl moieties of 1to 4 carbon atoms, provided that either R¹ or R² and either R³ or R⁴each represents a hydrogen atom, and a residue derived from the group ofthe above-mentioned formula by removal of OR¹, OR², OR³ or OR⁴ groupfrom one of its COOR¹, COOR², COOR³ and COOR⁴ groups (where the bond onthe carbon atom of the carbonyl group is attached to [SOD]), saidmonovalent copolymer group having an average molecular weight of 500 to200,000; and n represents an integer of 1 to 22 or 24 corresponding tothe number of said groups each derived from an amino group by removal ofone hydrogen atom in said [SOD] and/or a pharmaceutically acceptablesalt and a medium/higher fatty acid glyceride.
 3. The pharmaceuticalcomposition of claim 2 wherein said medium/higher fatty acid glycerideis one or a mixture of triglycerides of fatty acids containing 6 to 20carbon atoms.
 4. The pharmaceutical composition of claim 3 wherein saidmedium/higher fatty acid glyceride is caprylic acid triglyceride, capricacid triglyceride or a mixture thereof.
 5. A pharmaceutical compositionfor oral administration which comprises a superoxide dismutasederivative of the general formula

    [SOD][Z].sub.n

wherein [SOD] represents a superoxide dismutase having 1 to 22 or 24groups each derived from an amino group by removal of one hydrogen atomin lieu of amino groups; [Z] represents a monovalent copolymer group,constituting units of which are a group of the formula ##STR10## whereinR¹, R², R³ and R⁴ each represents a hydrogen atom or a residue derivedby removal of a hydroxyl group from an alkanol of 1 to 8 carbon atoms,an ethylene glycol monoalkyl ether containing an alkyl moiety of 1 to 4carbon atoms or a glycerin dialkyl ether containing alkyl moieties of 1to 4 carbon atoms, provided that either R¹ or R² and either R³ or R⁴each represents a hydrogen atom, and a residue derived from the group ofthe above-mentioned formula by removal of OR¹, OR², OR³ or OR⁴ groupfrom one of its COOR¹, COOR², COOR³ and COOR⁴ groups (where the bond onthe carbon atom of the carbonyl group is attached to [SOD]), saidmonovalent copolymer group having an average molecular weight of 500 to200,000; and n represents an integer of 1 to 22 or 24 corresponding tothe number of said groups each derived from an amino group by removal ofone hydrogen atom in said [SOD] and/or a pharmaceutically acceptablesalt thereof, a medium/higher fatty acid glyceride, and an amphiphilicagent and/or a lower alkanol.